Elucidation of the mechanism for differentiation of odontoblasts and mineralization of dentin

在牛成牙本质细胞无血清培养上清液中检测到活性明胶溶解金属蛋白酶、明胶酶A和明胶酶B。尤其是在培养的第21天左右，基质金属蛋白酶-2的活性突然出现，与成牙本质细胞突起的发育和碱性磷酸酶的丧失相一致。对这些成牙本质细胞的RT-PCR分析表明，在第15天到第21天之间，基质金属蛋白酶-2而不是基质金属蛋白酶-9的信息显著增加。这一体外观察表明，这些成牙本质细胞条件培养液中含有大量的基质金属蛋白酶-2，不仅降解胶原底物，而且还降解纯化的牙本质磷蛋白。我们还观察到，去磷酸化的牙本质磷蛋白在与基质金属蛋白酶-2有限的蛋白分解后成为酪蛋白激酶II的较好底物。这些结果支持我们的工作假设，即基质金属蛋白酶-2介导的蛋白分解过程是加速牙本质基质成熟过程的重要步骤，包括磷酸化和随后的矿化。作者和其他人提出了基质蛋白的细胞外磷酸化在骨和牙本质中的生物矿化中的意义(朱等人，Biochem。J.，323：637-643,1997；Mikuni-Takagaki等，J.bone Miner.研究报告，10：231-241,1995)。我们目前对MMP-2基因敲除小鼠的组织化学分析证实了这一观点，矿化组织、牙本质和骨的形成延迟。

In the serum-free culture medium of bovine odontoblasts, we detected active gelatinolytic metalloproteinases, MMP-2 and MMP-9 (gelatinase A and B). The activity of MMP-2, in particular, appeared suddenly around day 21 in the culture, coinciding with the development of odontoblastic cell processes and the loss of alkaline phosphatase. RT-PCR analysis of these odontoblasts demonstrated that messages of MMP-2 but not MMP-9 increased significantly between day 15 and day 21. This in vitro observation indicates that medium conditioned by these odontoblasts and containing significant amounts of MMP-2 mRNA, degrades not only the collagenous substrates but also purified dentin phosphophoryn as well.We have also observed that dephosphorylated dentin phosphoprotein beccmes a better substrate for casein kinase II after limited proteolysis with MMP-2. These results support our working hypothesis that MMP-2 mediated proteolytic processing is an important step in accelerating the process of dentin matrix maturation, which includes phosphorylation and subsequent mineralization. The authors and others have suggested the significance of extracellular phosphorylation of matrix proteins in biomineralization both in bone and in dentin (Zhu et al., Biochem. J., 323 : 637-643, 1997 ; Mikuni-Takagaki et al., J.Bone Miner. Res., 10 : 231-241, 1995). Our present histochemical analysis in MMP-2 knockout mice confirms this idea, with the the delayed formation of mineralized tissues, dentin and bone.